Crystal structure of human CD38 extracellular domain

Human CD38 is a multifunctional protein involved in diverse functions. As an enzyme, it is responsible for the synthesis of two Ca2+ messengers, cADPR and NAADP; as an antigen, it is involved in regulating cell adhesion, differentiation, and proliferation. Besides, CD38 is a marker of progression of HIV-1 infection and a negative prognostic marker of B-CLL. We have determined the crystal structure of the soluble extracellular domain of human CD38 to 1.9 A resolution. The enzyme's overall topology is similar to the related proteins CD157 and the Aplysia ADP-ribosyl cyclase, except with large structural changes at the two termini. The extended positively charged N terminus has lateral associations with the other CD38 molecule in the crystallographic asymmetric unit. The analysis of the CD38 substrate binding models revealed two key residues that may be critical in controlling CD38's multifunctionality of NAD hydrolysis, ADP-ribosyl cyclase, and cADPR hydrolysis activities.     

Crystal structure of a heat-resilient phytase from Aspergillus fumigatus, carrying a phosphorylated histidine

In order to understand the structural basis for the high thermostability of phytase from Aspergillus fumigatus, its crystal structure was determined at 1.5 A resolution. The overall fold resembles the structure of other phytase enzymes. Aspergillus niger phytase shares 66% sequence identity, however, it is much less heat-resistant. A superimposition of these two structures reveals some significant differences. In particular, substitutions with polar residues appear to remove repulsive ion pair interactions and instead form hydrogen bond interactions, which stabilize the enzyme; the formation of a C-terminal helical capping, induced by arginine residue substitutions also appears to be critical for the enzyme's ability to refold to its active form after denaturation at high temperature. The heat-resilient property of A.fumigatus phytase could be due to the improved stability of regions that are critical for the refolding of the protein; and a heat-resistant A.niger phytase may be achieved by mutating certain critical residues with the equivalent residues in A.fumigatus phytase. Six predicted N-glycosylation sites were observed to be glycosylated from the experimental electron density. Furthermore, the enzyme's catalytic residue His59 was found to be partly phosphorylated and thus showed a reaction intermediate, providing structural insight, which may help understand the catalytic mechanism of the acid phosphatase family. The trap of this catalytic intermediate confirms the two-step catalytic mechanism of the acid histidine phosphatase family.     

A conceptual model of the dynamics of the Syamozero Lake community

We present a conceptual mathematical model of the dynamics of a spatially heterogeneous population system whose prototype is the fish community of Lake Syamozero. Analysis of the solutions of this model is used to demonstrate that interactions between the predator and prey populations in two neighboring biotopes (the pelagic and coastal zones) may result in either undamped oscillations or steady states of the population sizes. The model population densities are of the same order of magnitude as the values obtained in long-term observations of the Syamozero biota. It is also demonstrated that the transition to steady states may be accompanied by long-term (dozens or hundreds of years) damped oscillations of the prey and predator population densities. Under natural conditions, long transitional periods may prevent fish communities from reaching stationary modes.     

Acidic residues at the active sites of CD38 and ADP-ribosyl cyclase determine nicotinic acid adenine dinucleotide phosphate (NAADP) synthesis and hydrolysis activities

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a novel metabolite of NADP that has now been established as a Ca(2+) messenger in many cellular systems. Its synthesis is catalyzed by multifunctional enzymes, CD38 and ADP-ribosyl cyclase (cyclase). The degradation pathway for NAADP is unknown and no enzyme that can specifically hydrolyze it has yet been identified. Here we show that CD38 can, in fact, hydrolyze NAADP to ADP-ribose 2'-phosphate. This activity was low at neutrality but greatly increased at acidic pH. This novel pH dependence suggests that the hydrolysis is determined by acidic residues at the active site. X-ray crystallography of the complex of CD38 with one of its substrates, NMN, showed that the nicotinamide moiety was in close contact with Glu(146) at 3.27 A and Asp(155) at 2.52 A. Changing Glu(146) to uncharged Gly and Ala, and Asp(155) to Gln and Asn, by site-directed mutagenesis indeed eliminated the strong pH dependence. Changing Asp(155) to Glu, in contrast, preserved the dependence. The specificity of the two acidic residues was further demonstrated by changing the adjacent Asp(147) to Val, which had minimal effect on the pH dependence. Crystallography confirmed that Asp(147) was situated and directed away from the bound substrate. Synthesis of NAADP catalyzed by CD38 is known to have strong preference for acidic pH, suggesting that Glu(146) and Asp(155) are also critical determinants. This was shown to be case by mutagensis. Likewise, using similar approaches, Glu(98) of the cyclase, which is equivalent to Glu(146) in CD38, was found to be responsible for controlling the pH dependence of NAADP synthesis by the cyclase. Based on these findings, a catalytic model is proposed.     

Structural Basis for Enzymatic Evolution from a Dedicated ADP-ribosyl Cyclase to a Multifunctional NAD Hydrolase

Cyclic ADP-ribose (cADPR) is a universal calcium messenger molecule that regulates many physiological processes. The production and degradation of cADPR are catalyzed by a family of related enzymes, including the ADP-ribosyl cyclase from Aplysia california (ADPRAC) and CD38 from human. Although ADPRC and CD38 share a common evolutionary ancestor, their enzymatic functions toward NAD and cADPR homeostasis have evolved divergently. Thus, ADPRC can only generate cADPR from NAD (cyclase), whereas CD38, in contrast, has multiple activities, i.e. in cADPR production and degradation, as well as NAD hydrolysis (NADase). In this study, we determined a number of ADPRC and CD38 structures bound with various nucleotides. From these complexes, we elucidated the structural features required for the cyclization (cyclase) reaction of ADPRC and the NADase reaction of CD38. Using the structural approach in combination with site-directed mutagenesis, we identified Phe-174 in ADPRC as a critical residue in directing the folding of the substrate during the cyclization reaction. Thus, a point mutation of Phe-174 to glycine can turn ADPRC from a cyclase toward an NADase. The equivalent residue in CD38, Thr-221, is shown to disfavor the cyclizing folding of the substrate, resulting in NADase being the dominant activity. The comprehensive structural comparison of CD38 and APDRC presented in this study thus provides insights into the structural determinants for the functional evolution from a cyclase to a hydrolase.Cyclic ADP-ribose (cADPR)³ is a calcium messenger ubiquitous in mammals as well as in invertebrates and plants and is responsible for regulating many physiological processes ranging from the simple function of calcium channel operation to the complex higher level organization of hormone secretion and autism (reviewed in Lee (1), Schuber and Lund (2), and Malavasi et al. (3)). The enzymatic production of cADPR from the substrate nicotinamide adenine dinucleotide (NAD) requires first the removal of the nicotinamide moiety followed by a cyclization reaction in which both ends of the remaining nucleotide are annealed (Fig. 1A). ADP-ribosyl cyclase (ADPRC) from Aplysia california was the first enzyme discovered to possess this function (cyclase) (4). Based on sequence homology (5), two human antigens, CD38 and CD157, were identified to also have the cyclase activity (6–8). However, different from ADPRC, which produces only cADPR from NAD, CD38/CD157 has evolved more like an NADase, producing mainly ADP-ribose (ADPR) from NAD, with cADPR being a minor product. The acquisition of the NADase and the cADPR hydrolysis activities of CD38 make it an important signaling enzyme in regulating NAD and cADPR homeostasis (9–11). Genetic analysis, as well as the conservation of sequence and disulfide bonds among these enzymes, establish that they all evolved from a common ancestor (12). Little is known of why this conserved family of enzymes has evolved divergently in their catalytic metabolism of NAD and cADPR.Open in a separate windowFIGURE 1.Schemes of cADPR formation and mechanistic analogs for substrate and product. A, the cyclization reaction producing cADPR from NAD is catalyzed by both ADPRC and CD38. The structural difference between cADPR and N1-cIDPR lies at the 6-position of purine ring (6-NH for cADPR; 6-O for N1-cIDPR). B, an analog of the substrate NAD, N(2F-A)D, is enzymatically converted to 2F-ADPR by ADPRC instead of cyclized to c(2F-A)DPR. The formation of cADPR from NAD requires the intramolecular attack of the reaction intermediate by the adenine N1 atom. The addition of a fluorine atom on the adjacent C2 atom of adenine prevents the cyclization from occurring. C, ara-2′F-NAD and ribo-2′F-NAD are analogs of NAD that inhibit the cyclization reaction by producing covalent adducts during the catalysis by CD38. Both analogs differ only in the orientation of their fluorine atoms at the 2′-position of the adenine ribose.ADPRC, however, is not solely a cyclase because it can also catalyze the hydrolysis of NMN into ribose-5-phosphate and nicotinamide (13, 14). The catalytic outcome of this novel enzyme is thus determined not by the enzyme alone but also by the specific interactions between the active site and a particular substrate. Consistently, using an NAD analog, N(2F-A)D, as substrate, Zhang et al. (15) showed that the hydrolase activity of ADPRC can be dominantly revealed, whereas its cyclase activity is suppressed beyond detection (Fig. 1B). Likewise, human CD38 can be converted to a ADPRC-like enzyme by mutation of a single residue, Glu-146, at the active site (16). In this study, we determined the structural determinants critical for the catalytic characteristics of ADPRC and CD38 by comparing the crystal structures of the complexes of ADPRC and CD38 bound with various catalytically revealing substrates and products (Fig. 1, A–C). The results identify residues Phe-174 in the cyclase and Thr-221 in CD38 as the main determinants for the cyclase and hydrolysis activities of the enzymes. All together, these structures provide insights into the structural requirements for functional evolution from a cyclase to a hydrolase.     

Resource availability and its role in development of invasion processes

The state of biotic communities inhabiting different water bodies is analyzed aiming to determination of conditions facilitating development of biological invasions. Mass-balance and dynamic models are used to reveal the factors ensuring the invasion of zebra mussel, Dreissena polymorpha, in Naroch Lakes (Byelorussia), and smelt, Osmerus eperlanus, in Lake Syamozero (Karelia). The results obtained indicate that for invasions leading to trophic chain lengthening, the main factor of successful invasion is the availability of resources that are not utilized by aboriginal species. Highest chances for successful invasion belong to invading species feeding on those trophic groups which contribute to the most extent to forming of 'excess' (i.e., available for the invader) production. It is shown that establishment of an alien species in a native community appears to be possible only when total amount of such 'excess' production is higher then some threshold value. Under conditions of spatio-temporal environmental heterogeneity, the timing factor, which determines the exact moment of an alien species intrusion, becomes important as well.     

Structure of a murine cytoplasmic serine hydroxymethyltransferase quinonoid ternary complex: evidence for asymmetric obligate dimers

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single carbon units for purine, thymidine, and methionine biosynthesis. The enzyme is a homotetramer comprising two obligate dimers and four pyridoxal phosphate-bound active sites. The mammalian enzyme is present in cells in both catalytically active and inactive forms. The inactive form is a ternary complex that results from the binding of glycine and 5-formyltetrahydrofolate polyglutamate, a slow tight-binding inhibitor. The crystal structure of a close analogue of the inactive form of murine cytoplasmic SHMT (cSHMT), lacking only the polyglutamate tail of the inhibitor, has been determined to 2.9 A resolution. This first structure of a ligand-bound mammalian SHMT allows identification of amino acid residues involved in substrate binding and catalysis. It also reveals that the two obligate dimers making up a tetramer are not equivalent; one can be described as "tight-binding" and the other as "loose-binding" for folate. Both active sites of the tight-binding dimer are occupied by 5-formyltetrahydrofolate (5-formylTHF), whose N5-formyl carbon is within 4 A of the glycine alpha-carbon of the glycine-pyridoxal phosphate complex; the complex appears to be primarily in its quinonoid form. In the loose-binding dimer, 5-formylTHF is present in only one of the active sites, and its N5-formyl carbon is 5 A from the glycine alpha-carbon. The pyridoxal phosphates appear to be primarily present as geminal diamine complexes, with bonds to both glycine and the active site lysine. This structure suggests that only two of the four catalytic sites on SHMT are catalytically competent and that the cSHMT-glycine-5-formylTHF ternary complex is an intermediate state analogue of the catalytic complex associated with serine and glycine interconversion.     

Stock status and current tendencies in the population dynamics of commercial fishes of Pskov-Chudskoe water body

Dynamics of eight commercial populations of fish from Pskov-Chudskoe Lake forming the main bulk (up to 80%) of annual catch was analyzed using an ADAPT-VPA model making it possible to reconstruct the dynamics of the total stock biomass and fishing mortality. The populations of five species (European smelt Coregonus albula, whitefish C. lavaretus maraenoides, perch Perca fluviatilis, roach Rutilus rutilus, ruffe Gymnocephalus cernuus) in the present period demonstrate a tendency for a decrease in biomass while the stocks of two species (bream Abramis brama and zander Sander lucioperca) beginning from the 1980s considerably increased. A particularly drastic (multifold) increase in biomass is recorded in zander population. The causes of the observed population changes are related mainly to ecosystemic transformations taking place in the water body; only with respect to two species-perch and roach-can one state that the decrease in stocks is determined in part by the fishery impact. The increase in zander population is, in all probability, the result of the combined effects of eutrophication forming favorable conditions for the feeding and survival of its juveniles and fishery that led to the decrease in the biomass of one of its main food competitors-perch. The drastic increase in zander numbers in turn promoted the decrease in the biomass of species being objects of its feeding.